RNA activation in ticks

RNA activation (RNAa) is a burgeoning area of research in which double-stranded RNAs (dsRNAs) or small activating RNAs mediate the upregulation of specific genes by targeting the promoter sequence and/or AU-rich elements in the 3′- untranslated region (3’-UTR) of mRNA molecules. So far, studies on the phenomenon have been limited to mammals, plants, bacteria, Caenorhabditis elegans, and recently, Aedes aegypti. However, it is yet to be applied in other arthropods, including ticks, despite the ubiquitous presence of argonaute 2 protein, which is an indispensable requirement for the formation of RNA-induced transcriptional activation complex to enable a dsRNA-mediated gene activation. In this study, we demonstrated for the first time the possible presence of RNAa phenomenon in the tick vector, Haemaphysalis longicornis (Asian longhorned tick). We targeted the 3ʹ-UTR of a novel endochitinase-like gene (HlemCHT) identified previously in H. longicornis eggs for dsRNA-mediated gene activation. Our results showed an increased gene expression in eggs of H. longicornis endochitinase-dsRNA-injected (dsHlemCHT) ticks on day-13 post-oviposition. Furthermore, we observed that eggs of dsHlemCHT ticks exhibited relatively early egg development and hatching, suggesting a dsRNA-mediated activation of the HlemCHT gene in the eggs. This is the first attempt to provide evidence of RNAa in ticks. Although further studies are required to elucidate the detailed mechanism by which RNAa occurs in ticks, the outcome of this study provides new opportunities for the use of RNAa as a gene overexpression tool in future studies on tick biology, to reduce the global burden of ticks and tick-borne diseases.


Results
Sequence analysis of the full-length HlemCHT. To obtain the full-length open reading frame (ORF) of the putative endochitinase-like gene (S03044-17L21 identified in the cDNA library, namely, HlemCHT), we employed the strategy summarised in Fig. 1a. Briefly, predicted exons and introns on the upstream sequence of the HlemCHT gene were retrieved from Chromosome 3 after a nucleotide BLAST search of a partial ORF sequence against both local (using BioEdit 7.2) and National Center for Biotechnology Information (NCBI) non-redundant (nr) protein databases of Haemaphysalis longicornis genome (GenBank accession number JAB-STR000000000). Forward and reverse primers (Red arrows) were then designed to target sequences before the predicted starting methionine, and sequences at about 150 bp downstream from the end of the partial ORF fragment respectively. Amplified cDNA was sequenced after cloning into a vector and the assembly of newly sequenced 5ʹ-cDNA fragment and already existing cDNA fragment into a contig sequence was done using CAP3 Sequence Assembly Program to obtain the full-length ORF. We finally obtained a cDNA of full length, 3768 bp (Fig. 1b), with an ORF of 1710 bp long. Analysis using the Compute pI/MW function of the Expasy web tool (www. expasy. org/ resou rces/ compu te-pi-mw) revealed that the ORF encoded 569 amino acid polypeptides, with a predicted molecular weight of 64.7 kDa. The 5ʹ-and 3ʹ-UTRs of the cDNA consisted of 215 bp and 1843 bp respectively (Fig. 1b). The full-length ORF sequences obtained in this study have been deposited in the DDBJ, EMBL, and GenBank databases. (Accession number LC744416).
Expression profile of HlemCHT transcripts in different life cycle and feeding stages. To  www.nature.com/scientificreports/ sion in the different life stages (12/15 days embryonated eggs, larvae. nymphs and adults) of H. longicornis parthenogenetic ticks at different feeding stages (unfed, partially fed and fully engorged) using reverse-transcription PCR (RT-PCR). The statutory expressed H. longicornis 40S Ribosomal S3a protein was used as an internal control gene, while a no template control served as a negative control. In our results, we observed HlemCHT to be solely present in the 12 and 15 dpo eggs (Fig. 2a), suggesting that the expression of HlemCHT is embryo-specific.
In addition, we investigated the expression profile of HlemCHT in tick eggs during embryogenesis. To do this, eggs from fully-engorged maternal ticks were divided into eight (1-, 4-, 7-, 10-, 13-, 16-, 19-and 21-days postinfection) groups, according to which, they were harvested and frozen. Total RNA was then extracted from each group of tick eggs (50-100 /group) and quantitative reverse-transcription PCR (RT-qPCR) analysis was performed. H. longicornis 40S ribosomal gene S3a was used to as a reference gene to normalize the HlemCHT gene expression data. From our results, the relative expression of HlemCHT increased significantly (P < 0.005) with embryo development until day 13 dpo, which recorded the highest expression (Fig. 2b)

Induction of RNAa in H. longicornis using dsRNA.
To investigate the potential occurrence of the RNAa phenomenon in eggs of H. longicornis, we targeted the 3ʹ-UTR of the HlemCHT gene because, among the many observations made concerning the genomic target site of saRNA, some have suggested that saRNAs demonstrate optimal function when they target sequences downstream from the 3′-UTR of the intended target gene 16 . Therefore, adult female ticks were injected with synthesized dsRNAs targeting either the 3ʹ-UTR of HlemCHT gene (dsHlemCHT) or a non-target control, Escherichia coli malE (Ec-malE) gene (dsEc-malE). RNA was then extracted from day 13 eggs derived from the dsRNA-injected maternal ticks because HlemCHT was observed to be highly expressed in tick eggs at 13 dpo (Fig. 2b), and as such, any changes in expression pattern, be it activation or suppression, would be clearly shown. The expression of HlemCHT was quantified by RT- www.nature.com/scientificreports/ qPCR analysis. In our results, targeting the 3ʹ-UTR, showed a 19 ± 8.20-fold significant (P = 0.0145) increase in HlemCHT expression in dsHlemCHT-derived eggs relative to dsEc-malE-derived eggs (Fig. 3a). In addition, morphological examination of the dsHlemCHT-derived eggs at 13 dpo, showed a normal phenotype characteristic of stage 12 embryo described in previous studies 44,45 with evidence of limb formation, whitish strands of malpighian tubules and a distinctly formed bright white hindgut, similar to dsEc-malE-derived eggs (Fig. 3b). Moreover, dsHlemCHT-derived eggs were observed to begin hatching within 20 days post-oviposition, about 24 h earlier than dsEc-malE -derived eggs (Fig. 3c). Furthermore, at 21 days post-oviposition, when dsEc-malEderived eggs began hatching, the hatching rate was 32.9 ± 32.6%. In contrast, the hatching rate for dsHlemCHTderived eggs was relatively higher at 52.95 ± 13.50%, although not significant (P = 0.5066) (Fig. 3c,d). On the other hand, when the ORF of the same gene was targeted, the relative expression of HlemCHT was observed to be 0.09 ± 0.02 in dsHlemCHT-derived eggs as opposed to 1.00 ± 0.04 in dsEc-malE-derived eggs, suggesting a significant (P = 0.0001) downregulation of the gene in dsHlemCHT-derived eggs at 13 dpo (Fig. 4a). Interestingly, phenotypic observation of day 13 dsHlemCHT-derived eggs also revealed a normal phenotype as observed when the 3ʹ-UTR was targeted, with no difference observed between the dsEc-malE-derived and dsHlemCHTderived eggs (Fig. 4b). Consequently, hatching of dsRNA-derived eggs begun on day 22 post-oviposition, with no significant (P = 0.5306) difference observed between hatching rates of dsHlemCHT-derived eggs (27.73 ± 4.00%) and dsEc-malE-derived eggs (32.26 ± 5.51%) (Fig. 4c,d). These results, therefore, indicate the importance of the 3ʹ-UTR as an essential target for dsRNA-mediated activation of the HlemCHT gene in tick eggs.

Discussion
RNAa is the targeted upregulation of a specific gene 21 . The use of RNAa was limited to only a few major mammalian models 3,11,13,46 , but it has now been reported in other organisms such as plants, bacteria, C. elegans, and recently, mosquito arthropods. It is currently being explored as a potential tool for the specific activation of genes, towards the development of novel therapeutics for undruggable disease and vector control 14,17 .
In the present study, we targeted the 3ʹ-UTR of a novel endochitinase-like gene (HlemCHT), in H. longicornis eggs for dsRNA-mediated gene activation and showed an increased expression of the gene on day 13 postoviposition. In a simultaneous experiment, when we targeted sequences within the ORF of the same gene, we observed a downregulation of the gene (Fig. 4), suggesting the possibility of a dsRNA-mediated gene activation, which may be based on the association of the dsRNA (saRNA) with the 3ʹ-UTR of the gene.
Different dsRNA-mediated gene activation mechanisms have been proposed to involve the 3ʹ-UTR. For instance, it has been suggested that the saRNAs that target the 3ʹ-UTR trigger a looping mechanism bringing www.nature.com/scientificreports/  www.nature.com/scientificreports/ the 3′-terminus and promoter into proximity to allow the recruitment of additional proteins and modulation of promoter activity 16 . It has also been proposed that saRNA molecules may target AU-rich elements in 3ʹ-UTR of mRNA molecules and induce gene expression at the transcriptional level. For example, two studies showed miR-369-3 to positively regulate mRNA translation by targeting AU-rich elements in 3′-UTRs in stressed cells 47 . However, in this study, characteristic AU-rich elements were not found in the 3′-UTR of HlemCHT, which suggests that the gene activation observed in our study may probably be due to a looping mechanism triggered by saRNAs targeting the 3′-UTR, and bringing the 3′ terminus and promoter into proximity for the activation of the gene, leading to a 19-fold increase in gene expression. Further studies are however required to elucidate the exact mechanism of RNAa in ticks. This study also established a positive relationship between dsRNA-mediated gene expression and egg hatching, concerning hatching start time and percentage of hatched eggs. We observed that eggs of dsHlemCHT began www.nature.com/scientificreports/ hatching about 24 h earlier than control eggs, as well as a slightly higher percentage of hatched eggs by 21 dpo, suggesting that the possible dsRNA-mediated gene activation of HlemCHT observed on day 13, drove the early hatch of dsHlemCHT -derived eggs. Indeed, several reports on the role of chitinase in eggs of arthropods and nematodes, have indicated that chitinase (-like) genes are vital for successful egg development and hatching [48][49][50][51][52][53] . For example, knockdown of Tribolium (flour beetle) chitinase-like gene, TcCHT10 in adult females, resulted in none of the eggs hatching, despite the presence of fully developed embryos in these eggs 52 . In our study, a single injection of dsRNA targeting the ORF, into unfed adult female ticks resulted in no phenotypic changes, despite the downregulation of the HlemCHT gene observed (Fig. 4). However, a repeated introduction (once daily for 3 consecutive days) of dsRNA into fully-engorged adult female ticks before the start of oviposition resulted in a downregulation of the gene and the subsequent delay or reduction of hatching rate of H. longicornis eggs. The results for this modified RNAi method will be reported in another article being prepared currently.
Overall, this study's results provide some evidence of the presence of RNAa phenomenon in the tick vector, H. longicornis for the first time. More studies are however required to provide further insight into the detailed mechanisms by which the expression of genes is activated by dsRNA (saRNAs) in ticks. Currently, no established gene manipulation technology permits stable gene knock-ins for applications such as gene replacement and over-expression in ticks 41 . Therefore, the discovery of RNAa phenomenon in ticks has the potential to provide new technology for the application of RNAa in the over-expression of both endogenous and exogenous genes in ticks. For this purpose, we will investigate other H. longicornis genes for the established phenomenon of RNAa in subsequent studies. Furthermore, reports of RNAa in Ae. aegypti 17 and H. longicornis (this study), show the potential of saRNAs 40 , considering their small size, versatility, and safety, to reverse phenomena, such as the emergence of acaricide/insecticide resistance due to the downregulation of critical genes 54,55 . Finally, this report will hopefully provide new opportunities, based on the usefulness of RNAa as a tool for future research in arthropod biology, to modulate gene responses, to restrict the expansion of tick populations and transmission of pathogens to reduce the global burden of ticks and tick-borne diseases.

Materials and methods
Ticks and animals. The parthenogenetic Okayama strain of the ixodid tick H. longicornis bred by feeding on mice as described previously 56 , was maintained at the Department of Parasitic and Tropical Medicine, Kitasato University School of Medicine (KUSM), Sagamihara, Kanagawa, Japan. Its ability to reproduce parthenogenetically by feeding on the blood of several hosts including mice makes it easier to maintain in the lab. Therefore has become an ideal model tick species for tick experiments in many labs globally as can be seen in various publications 27,37,57-66 . Fifteen-week-old BALB/c CrSlc mice (SLC Japan, Shizuoka, Japan) were adapted to standard animal husbandry conditions (25 °C, 60% Relative Humidity (RH)) for 7 days, and subsequently to tick maintenance condition (25 °C, 70% RH) for 2 days before the experiment. Cloning and sequencing of full-length HlemCHT. The plasmid (pCR-Blunt II-TOPO/S03044-17L21) containing partial cDNA encoding the HlemCHT transcript was first isolated from a previously established cDNA library 43 . A BLASTn search of the partial sequence of the 5ʹ-terminal (ATG ACG TAT GAT CTG CGT GGG AAC TGG GCT GGC TTT ACGG) of this cDNA was performed locally against Haemaphysalis longicornis genome database (GenBank accession number JABSTR000000000) with the p-value of < 10 -6 by using BioEdit 7.2 (https:// bioed it. softw are. infor mer. com/7. 2/). According to this analysis, the partial sequence was identified in Chromosome No.3 with an expected value of 1.00E−12 and an identity of 97%. By using this partial genome sequence, the BLASTN program on the web tool (https:// blast. ncbi. nlm. nih. gov/ Blast. cgi? PAGE_ TYPE= Blast Search) was done again against the NCBI nr database and the predicted HlemCHT gene (GenBank accession number currently not available due to ongoing updates of genome annotations) located on the chromosome No. 3 of H. longicornis was identified. According to the previous (11th February 2022) annotated information, the upstream sequence including predicted exons and introns was retrieved to design the forward primer on the most upstream region before starting methionine. Reverse primer was also designed at about 150 bp downstream of the edge of known cDNA fragment. Both forward and reverse primers on the 5ʹ-UTR were designed to amplify the unknown fragment flanked by the starting methionine and the partial cDNA sequence. Primers used in this study were purchased from Eurofins Genomics K.K., Tokyo, Japan, and are listed in Table 1 www.nature.com/scientificreports/ Kit (Invitrogen by ThermoFisher Scientific K.K., Tokyo, Japan), after which samples were sequenced by Sanger technology in an independent external company (Eurofins Genomics K.K., Tokyo, Japan). Starting methionine and 5'UTR sequence were confirmed accordingly. The newly sequenced 5ʹ-cDNA fragment and already existing cDNA fragment were assembled into a contig sequence using CAP3 Sequence Assembly Program on the web tool (https:// doua. prabi. fr/ softw are/ cap3) 69 . Full-length ORF predicted to encode this novel endochitinaselike protein was isolated and confirmed from another set of RT-PCR with the Full HlemCHT ORF primer set (Table 1) and sequence analysis at the company (Eurofins Genomics K.K., Tokyo, Japan). The strategy for cloning and sequencing of full-length HlemCHT is illustrated in Fig. 1a.

Expression pattern of HlemCHT in different life cycle and feeding stages.
To determine the normal expression of HlemCHT in different developmental and feeding stages of H. longicornis, reverse transcription-polymerase chain reaction (RT-PCR) was employed. To do that, larval (150), nymphal(100) and adult (7) stages of H. longicornis ticks were allowed to feed on the shaven back of BALB/c mice (3 mice per each developmental stage). At the beginning of the tick expansion period (3-5 days after tick attachment), About 50 partially engorged larvae, 50 partially engorged nymphs and 5 partially engorged adult female ticks, were detached carefully with fine tip forceps without breaking the hypostome (in the case of the adults) or gently passing a soft-bristled paint brush over the attached ticks to detach them (in the case of larvae and nymphs). The rest of the ticks were allowed to fully engorge and drop off by themselves. Two fully engorged adult ticks were allowed to lay eggs under the suitable temperature of 25 °C and > 70% humidity. Eggs laid by each engorged tick were further divided into two groups (50-100/group) and incubated for either 12 or 15 days post oviposition. Either tick developmental stages (unfed, partially fed and fully engorged (after egg laying)) or egg samples collected at their respective times were immediately flash frozen in liquid nitrogen and stored at − 80 °C until used. For RNA extraction, frozen samples were ground to a fine powder, using a mortar and pestle kept in liquid nitrogen. Total RNA was isolated from the resulting powder with the RNeasy mini kit (Qiagen, Valencia, CA, USA) according to the manufacturer's instructions. The quantity and integrity of the RNA was measured spectrophotometrically by ultraviolet light absorbance using NanoDrop™ One Microvolume UV-Vis Spectrophotometer (Thermo Scientific, Rockford, IL, USA) and stored at − 80 °C until use. Complementary DNA (cDNA) was synthesized www.nature.com/scientificreports/ from about 150 ng total RNA extracted from the tick/egg samples using ReverTrace™ qPCR RT Master Mix with gDNA remover (Toyobo, Japan) kit according to manufacturer's instructions. Polymerace chain reactions (PCRs) were performed to amplify a 110 bp region of HlemCHT and a 192 bp region of 40S Ribosomal protein S3a using primers purchased from Eurofins Genomics K.K., Tokyo, Japan. Primers are shown in Table 1. The reaction mixtures for PCR were prepared using the KOD Plus Neo ™ PCR kit (Toyobo, Japan) and band amplification was done using a Bio-Rad T100™ Thermal Cycler, with the following cycling conditions; initial denaturation at 94 °C for 2 min, followed by 35 cycles of denaturation at 98 °C for 10 secs, annealing at 66 °C for 30 secs (for both genes) and extension at 68 °C for 10 secs, with a final extension performed at 68 °C for 1 min. The PCR products were separated by a 1.5% agarose gel electrophoresis and stained with ethidium bromide for viewing.
Expression pattern of HlemCHT in tick eggs during embryo development. By the same method of blood feeding and egg laying described above, eggs were collected from two different fully engorged maternal ticks, and each set was divided into eight (1-, 4-, 7-, 10-, 13-, 16-, 19-and 21-days post-infection) groups, according to which, eggs were harvested and frozen. Total RNA from tick eggs (about 50-100/group) was extracted, followed by subsequent cDNA synthesis as have already been described in this study. RT-qPCR analysis was then performed using TB Green® Premix DimerEraser™ kit (Takara, Japan) according to the manufacturer's instructions with the same primer sets (Table 1) already described for either HlemCHT or H. longicornis 40 s ribosomal protein S3a to determine their expression levels. Each experiment was done with two different RNA samples per each group and were run in duplicates. The experiment was repeated three times, and the data were analyzed with the 2 −ΔΔCt method 70 using Microsoft Excel version 2019 and expressed as mean ± standard deviation. Statistical significance was determined using an unpaired two-tailed student's t-test, with a value of P < 0.05 considered significant after the normal distribution and homogeneity of variance were confirmed by Kolmogorov-Smirnov and F-tests respectively.

Double-stranded RNA (dsRNA) synthesis.
To investigate the occurrence of RNAa in ticks, the 3ʹ-UTR of the HlemCHT gene was targeted. A template partial-length cDNA fragment at the 3ʹ-UTR of the HlemCHT transcript (497 bp) was therefore amplified from a pCR-Blunt II-TOPO/S03044-17L21 plasmid by PCR, using primer sets flanked by a T7 promoter and gene-specific primer sets (Table 1) which were designed using Primer 3 (v. 0.4.0) program (https:// bioin fo. ut. ee/ prime r3-0. 4.0/) and purchased from Eurofins Genomics K.K., Tokyo, Japan. To provide further evidence that the association of dsRNA to the 3ʹ-UTR is vital for the occurrence of RNAa, we also synthesized dsRNA targeting the ORF in a parallel experiment. A template partial-length cDNA fragment within the ORF of the HlemCHT transcript (586 bp) was therefore amplified from the same plasmid using T7 promoter-flanked primers, as well as gene-specific primer sets designed and purchased as already described ( Table 1). As a negative control (also a non-target control), dsRNA targeting Escherichia coli malE (Ec-malE) was also synthesized. Ec-malE is absent in ticks and dsRNA targeting Ec-malE has been shown in several studies [61][62][63]71 not to affect normal tick physiology, including egg laying and the normal expression of tick-specific genes. Moreover, it has been shown to give comparable results to a no injection (non-manipulated) control in most of the studies referred to above. Briefly, the Ec-malE DNA fragment (848 bp) was first amplified from E. coli using primers (Table 1) designed and purchased as already described. The amplified fragments were then cloned into the pCR-Blunt II-TOPO vector as earlier described. Positive colonies were sub-cultured and plasmid purification was also done as already described. A template DNA fragment of the Ec-malE gene (577 bp) was then amplified from the pCR-Blunt II-TOPO/malE plasmid by PCR, using T7 promoter-flanked primers, as well as gene-specific primer sets designed and purchased as already described ( Table 1). The amplification was performed using a PCR program of 94 °C for 2 min, 35 cycles of 98 °C for 30 s, either 66 °C (HlemCHT/3ʹ-UTR), 66 °C (HlemCHT/ORF) or 67 °C (Ec-malE) for 30 s, and 68 °C for 30 s, followed by elongation for 1 min at 68 °C. The PCR products were purified from agarose gel using VIOGENE Gel/PCR DNA isolation system Extraction Kit (VIOGENE BIOTEK, TAIWAN) according to the manufacturer's instructions. DsRNAs of HlemCHT (3ʹ-UTR), HlemCHT (ORF) and Ec-malE were synthesized by in vitro transcription using the T7 RiboMAX™ Express RNAi System (Promega, Madison, WI, USA) according to the manufacturer's instructions. The purity of synthesized dsRNAs was checked by running 1 µL of diluted dsRNA (1:10, 1:100, and 1:1000 with Nuclease-Free water) in a 1.5% agarose gel electrophoresis. Concentrations of dsRNAs were also measured by ultraviolet light absorbance using NanoDrop™ One Microvolume UV-Vis Spectrophotometer (Thermo Scientific, Rockford, IL, USA).

Microinjection of dsRNA into adult female ticks.
A volume of 0.5 μl of 2 μg/μl either HlemCHT (3ʹ-UTR), HlemCHT (ORF) or Ec-malE dsRNA was injected into the hemocoel from the fourth left coxa of unfed adult female H. longicornis ticks (10 ticks per each treatment) with a glass needle generated by a micropipette PC-10 puller (Narishige International, NY, USA) using an IM-11-2 pneumatic microinjector (Narishige International, NY, USA). Injected ticks were left for 18 h at 25 °C in a moist chamber. After 18 h, the dsRNA-injected ticks (dsHlemCHT (3ʹ-UTR), dsHlemCHT (ORF) and dsEc-malE) were observed for any mortality due to traumatic injury resulting from injection. Live ticks within each treatment group were then placed on a mouse to blood feed as earlier described. After 3-4 days of tick attachment, partially fed ticks were removed, leaving 2 ticks on each mouse for optimal blood feeding and engorgement. Each engorged tick was allowed to lay eggs after detachment from the host mouse.
Maintenance and monitoring of dsRNA-injected ticks' eggs. Eggs laid daily by each of the engorged ticks were kept separately in the wells of 24-well plates under humidified conditions at 25 °C. Some of the egg samples were allowed to develop and hatch, while others were collected on day 13 post-oviposition for con- www.nature.com/scientificreports/